Emergence of multidrug-resistant Bacillus spp. derived from animal feed, food and human diarrhea in South-Eastern Bangladesh

Background Antimicrobial resistance poses a huge risk to human health worldwide, while Bangladesh is confronting the most severe challenge between the food supply and the huge consumption of antibiotics annually. More importantly, probiotics containing Bacillus spp. are claimed to be an alternative to antimicrobial stewardship programs. However, their antibiotic resistance remains elusive. Thus, we employed the antimicrobial susceptibility test and PCR to assess the prevalence of resistance, including multidrug resistance (MDR) and resito-genotyping of isolated Bacillus spp. Results The phenotypic profile showed that Bacillus spp. were 100% sensitive to gentamicin (2 µg/mL), whereas lowered sensitivity to levofloxacin (67.8%, 0.5–1 µg/mL), ciprofloxacin (62.3%, 0.5–1 µg/mL), clindamycin (52.2%, 0.25–0.5 µg/mL), amoxicillin-clavulanic acid (37.6%, 0.06 µg/mL), azithromycin (33.4%, 1–2 µg/mL), tetracycline (25.6%, 2–4 µg/mL), nitrofurantoin (21.1%, 16–32 µg/mL), co-trimoxazole (19.2%, 2 µg/mL), and erythromycin (18.8%, 0.25–0.5 µg/mL). The strains were completely resistant to penicillin, amoxicillin-clavulanic acid, cefixime, ceftriaxone, vancomycin, and co-trimoxazole, and a species-specific trend was seen in both phenotypic and genotypic resistance patterns. Genotypic resistance indicated prevalence of the bla1 (71.5%), tetA (33%), erm1 (27%), blaTEM (13.1%), blaCTX-M-1/blaCTX-M-2 /sul1 (10.1%), blaSHV (9.6%), and qnrS (4.1%) genes. The β-lactamase resistance gene bla1 was found in all penicillin-resistant (MIC ≥ 32 µg/mL) Bacillus spp. One hundred ninety-one isolates (89.6%) were MDR, with 100% from diarrhea, 90.3% from food, and 88.7% from animal feed. Conclusion Based on the MIC value and profile analysis of antibiotic resistance genes, this is the first study that Bacillus spp. antimicrobial susceptibilities have been identified in Bangladesh, and our study will shed light on the adverse effects of feed-borne Bacillus spp. emerging from animal feed to the food chain. A comprehensive investigation is urgently needed by policymakers on tolerance limits and harmful effects in the animal industry. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-024-03199-3.


Background
Antimicrobial resistance (AMR) is a serious, multifaceted, and complicated healthcare concern worldwide that impacts people, animals, and the environment, resulting in harder-to-treat infections and even death.The "One Health" approach, which incorporates public health and veterinary regulators, the food and agriculture industry, financiers, environmentalists, and customers, is highlighted in the WHO-led Global Action Plan on Antimicrobial Resistance [1,2].AMR develops naturally over time, generally through genetic mutations that can transmit from one generation to another or between humans and animals via animal-sourced food.A variety of strategies, including target defence, target replacement, detoxification, and suppression of cellular antibiotic deposition, are used by bacteria to develop antimicrobial resistance (AMR) [2][3][4].Although not all resistant bacteria produce diseases, they may initiate the manifestation of a disease or spread the gene encoding AMR to new bacterial pathogens in favorable environments [4].Consequently, improper and abusive antibiotics might contribute to the development of different drug-resistant bacteria and can disperse antibiotic residues from various settings throughout the food supply chain, acting as a reservoir and propagation matrix for AMR with the potential for antibiotic-resistant gene (ARG) to cross the animal-tohuman microbes due to bacterial contamination [5][6][7].The transfer of ARGs is a common way that ABR spreads.Once resistant genes are transmitted by plasmids, transposons, or integrons, dispersion is quick, and horizontal gene transfer across bacteria is frequent.Hitherto, it was thought that this type of genetic exchange only occurred among the same bacterial species.Nevertheless, the transmission of ARGs among phylogenetically distinct bacterial clusters, particularly across gram-positive and gram-negative bacteria, has now been proven in natural habitats [8].
Bacillus spp.has long been used as probiotics in human, veterinary, aquaculture, plant, and environmental applications, either directly as microbial food or as food additives heavily contaminated in animal feed and food chains, making a major financial burden for livestock producers and a potential threat to public health [6,[9][10][11].B. cereus-caused foodborne diseases are classified into diarrheal (toxico-infections) and emetic (intoxications) syndromes resulting from the formation of several toxins (enterotoxins such as nhe, hbl, cytK, entFM, BceT, HlyII; emetic toxins ces), which occur globally and are becoming a serious challenge [7,12].B. cereus exacerbates severe diarrhea and malnutrition in chickens and ducks by causing gizzard erosion and ulceration (GEU) and facilitating recurrent bacterial infections in the lungs by disrupting the gastrointestinal tract and following lung hemorrhagic lesions [13][14][15][16].More interestingly, B. cereus was reported to induce non-gastrointestinal diseases, including bacteremia, septicemia, endophthalmitis, meningitis, endocarditis, urinary tract infections, and lung infections.Furthermore, B. cereus may lead to serious health effects, especially in newborn infants and immunosuppressed individuals [7,12].
Bangladesh, with a significant level of AMR and multidrug-resistant (MDR) bacteria against drugs indicated for use in both animals and people, confronts a local and worldwide hazard [3,[17][18][19].However, B. cereus is resistant to numerous antibiotics, posing a global issue [20].To inhibit the spread of AMR, it is essential to evaluate Bacillus spp.and their AMR profile.Some strains of Bacillus spp.are becoming increasingly resistant to antibiotics, allowing for the acquisition and emergence of new AMR strains.In our prior report, 39% of Bacillus spp.from animal feed and animal-based foods at a contamination level > 10 5 CFU/g carried 80%, 71%, 55%, and 33% of the entFM, cytK, nheABC, and hblACD enterotoxin genes, respectively, and foodborne Bacillus spp.caused 4.5% of human diarrhea cases in south-eastern Bangladesh [15].There is a lack of scientific data on the AMR in the livestock sector in Bangladesh.According to available report, resistant bacteria such as E. coli, Salmonella spp., Klebsiella spp., Pseudomonas spp., Staphylococcus spp., and Vibrio spp., were commonly detected in poultry, dairy cattle, raw milk, farm surroundings, and fish items [21,22].Nevertheless, there is a dearth of comprehensive data regarding the antibiotic resistance patterns of Bacillus spp. in the human, animal, and environmental sectors in Bangladesh.Thus, to fillin this knowledge gap, this work aimed to determine the prevalence of resistance, particularly MDR, and the correlated genetic factors in isolated Bacillus spp.In the present study, we focused on whether animal feed, food, and human stool could harbor MDR strains and disseminate them through the food supply chain.

Bacterial isolates
The isolates tested in this investigation were chosen from our prior work [15]

Pearson correlation coefficients (ρ) for pairs of antibiotics to assess ABR Bacillus isolates
Bivariate analysis showed a highly significant association (p = < 0.001-0.000)between the resistance patterns of TET and PG, EM/NIT and CFM, AZM/CM and AMC, NIT/CFM/CTR/ VAN 4).

Discussion
In the current study, the isolated Bacillus strains were sensitive to GEN, CIP, LEV, CM, AMC, TET, EM, AZM, NIT, and CMX, which was consistent with the previous reports [4,5,7,9,12,20,23,24].However, there were species-specific sensitivities of the Bacillus strains to CIP, LEV, CM, TET, EM, AZM, and NIT at various doses.Regarding the resistant pattern, diarrheal strains were completely resistant to PG, AMC, CFM, CTR, VAN, and CMX, whereas animal feed-borne strains were generally resistant to PG, AMC, CFM, VAN, and CMX, and foodoriginated strains were generally resistant to PG, CFM, CTR, and CMX.As for different species, B. cereus, B. thuringiensis, and B. licheniformis strains were all completely resistant to the beta-lactam antibiotics of PG and  The B. cereus strains were generally sensitive to GN, CIP, and LEV, while intermediately sensitive to AZM, CM, and TET.In contrast, they were generally resistant to PG, CFM, CTR, CFM, VAN, EM, AMC, NIT, and TET.This result agreed with earlier findings [4,7,20,23,24,[26][27][28], which were isolated from rice, cereals, chicken meat, fresh vegetables, edible fungi, powdered milk, foodstuffs, human stool, and clinical samples.The isolated B. subtilis was generally sensitive to GEN, LEV, CM, and CIP; intermediately sensitive to EM, TET, AZM, and NIT; and generally resistant to CFM, PG, CMX, CTR, VAN, and AMC.However, this bacteria strain detected in bread, powdered milk, soil, and shrimp culture ponds showed sensitivity to GEN, VAN, CM, EM, TET, and CMX while being resistant to PG, ampicillin, cefpodoxime, and cefepime [4,9,29,30].These two dominant species had species-specific responses to AMC, AZM, EM, TET, NIT, and LEV.The main factor might be large abuse, which prolongs time.The high sensitivity to GEN, CIP, and LEV could be attributed to the limited administration of CIP and LEV, while GEN is not absorbed via oral application.Consequently, CTR, CFM, AMC, CMX, and NIT were largely abused in the animal industry and added frequently against infectious diseases.On the other hand, antibiotic regulation contributes to AMR.In several countries, CIP and LEV are prohibited for use on animals due to human drugs.
It is worth mentioning that 89.6% of Bacillus isolates were MDR, with 100% of isolates from diarrhea, 90.3% from food, and 88.7% from animal feed exhibiting the MDR pattern.These findings were consistent with other reports that found MDR Bacillus spp. in several sources, including food [4,5,7,31,32].In our investigation, 100% B. cereus and 91.8% B. subtilis isolates yielded MAR indexes > 0.2, indicating plasmid-mediated resistance and a significant risk of contamination.This implies a high inclination and trend for antibiotic resistance among the MDR bacterial isolates [33].In Bangladesh, MDRs of E. coli, Salmonella spp., Campylobacter spp., and Enterobacter spp.were detected in livestock populations due to contamination of animal-derived food and food products [3].However, MDR in Bacillus spp.has not been reported.Our data indicated Bacillus strains might produce extended-spectrum beta-lactamase (ESBL) and resistance to third-generation cephalosporins (CFM and CTR), macrolide (EM), tetracycline, second-and thirdgeneration quinolones (CIP and LEV), and sulfonamides (CMX).
In this study, genes encoding β-lactamase (bla1, bla TEM , bla CTX-M-1 , bla CTX-M-2 , bla SHV ), fluroquinolone (qnrS), sulfonamide (sul1), tetracycline (tetA), and macrolide Fig. 2 Distribution of antibiotic resistance genes of Bacillus spp.isolated from animal feed, food and diarrheal cases in Bangladesh.The numerical value displayed above each bar shows the positive rate associated with the respective antibiotic resistance genes Fig. 3 Species distribution of antibiotic resistance gene of Bacillus spp.isolated from animal feed, food and diarrheal cases in Bangladesh.The numerical value displayed above each specific color bar shows the positive rate associated with antibiotic resistance genes of the respective Bacillus strains Fig. 4 Sample wise distribution of antibiotic resistance gene of Bacillus spp.isolated from animal feed, food and diarrheal cases in Bangladesh.The numerical value displayed above each specific color bar shows the positive rate associated with respective antibiotic resistance genes in animal feed, food and diarrhea (erm1) were derived from animal feed, food, and diarrhea.The most commonly observed β-lactamase genes were bla1 than bla TEM , bla CTX-M-1 , bla CTX-M-2 , and bla SHV among Bacillus strains.Similar detection of the bla1 gene in Bacillus strains was reported by other isolates from chicken meat, meat products, human stool, food, and the CFM-EM-TET-CM-CMX environment [27,34,35].However, some of our findings were inconsistent with the previous studies [23,36,37].They observed a higher prevalence of bla TEM , bla CTX-M-1 , bla CTX-M-2 , and bla SHV in waste water, clinical samples, and food samples.The second dominant gene was the tetA gene in our study, which was consistent with prior studies [8,18,25], but other authors reported a higher occurrence [28,35].The prevalence of the erythromycin-resistant gene, erm1, was low compared to earlier reports [9,25,35,37].Furthermore, our study revealed a low prevalence of sulfonamide, sul1 and quinolone, and qnrS resistance genes.The sul1 gene in Bacillus strains isolated from aquaculture ponds was lower than our report [1], whereas the sul1 gene in wastewater was higher than our report [37].Bacillus spp.are often used as food microbial additives and spread ARGs by horizontal transfer of plasmids, leading to the failure of antibiotic treatment and dramatically altering their phenotypes [4,5,38].The Bacillus anthrax-associated plasmids pXO1 and pXO2 have been detected in certain B. cereus strains with pathogenic potential resembling B. anthracis [39].For instance, atypical B. cereus strains such as B. cereus G9241, B. cereus biovar anthracis CA, Bcbva, and Bcbvalike strain BC-AK were linked to anthrax-like disease in mammals, livestock, and humans in the United States, China, and West Africa, implying that it may be widespread [40].The highly efficient mobilization capacities and horizontal gene transfer may pose a serious threat to gene circulation, particularly ARGs.Probiotic Bacillus spp.have already been connected to clinical infections, as well as β-lactams, aminoglycosides, macrolides, chloramphenicol, tetracycline, and erythromycin resistance genes, which may contribute to the spread of ABR in animal microbiota and the possible transmission of ARG to humans [10].Nevertheless, abusive antibiotic use can transmit antibiotic residues in foods derived from animals, like milk, meat, and eggs, as well as in the environment [6].As a potential driver of both genes and bacteria resistant to antibiotics, the ARG may be further transmitted to people directly through the food chain [10].
The high resistance of Bacillus strains to CFM, CTR, AMC, and PG might be caused by β-lactamase and the presence of ABC (ATP binding cassette) efflux transporters from B. subtilis that are tolerant to lincosamide [25,26].The different antibiotic mechanisms might be associated with inherent resistance, built-up resistance, gene modification, and DNA transfer that aid in bacterial survival by manipulating the penicillin binding protein (PBP), enzymatic blockage, porin mutations, and efflux pumps [6].The current study revealed that Bacillus isolates primarily carried the β-lactamase resistance genes bla1, bla TEM , bla CTX-M-1 , bla CTX-M-2 , bla SHV , tetracycline resistance gene tetA, and erythromycin resistance gene erm1, respectively.It was confirmed that certain ARG classes could be acquired by the majority of antibiotics and evaluated in various Bacillus strains.According to resistant gene distribution, 10 distinct ARG patterns were detected in the isolates.The association of the above β-lactamase and other antibiotic genes within the same isolate has been reported [9,23,28,36,37].However, the most common associations were bla1 + erm1, bla1 + tetA, bla1 + bla TEM , bla TEM + tetA, and bla CTX-M-1 + tetA (Table S2).This occurrence indicates a greater spread of β-lactamase, tetracycline, and erythromycin genes, most likely owing to a genetic component in their mobilization as well as the horizontal transfer of ABR determinants between Bacillus strains or from other bacteria into Bacillus spp.[7,8,36].
In our prior study, we revealed a high Bacillus spp.contamination level with significant toxigenic potential in several resources, including animal feed, animal-derived goods, and regular food items [15], where 90.3% of isolates displayed > 0.2 MAR index, indicating a high-risk source of contamination [41].The presence of MDR and MAR Bacillus in animal feed, food, and diarrhea indicated that the abuse of antibiotics poses a severe public health hazard by transmitting AMR to people through the food supply chain.
There is a dearth of research data on the transmission of ARGs and the AMR of Bacillus strains in Bangladesh.According to a review report, the emergence of AMR is mainly attributed to antibiotic misuse or overuse by broiler (> 60%) and layer (94.6%) farmers as over-the-counter medication and failure to maintain the withdrawal period [3].Empirical data suggests that antibiotic residues against Bacillus spp.exist in the liver and kidney as well as in commercially available feed in Bangladesh, acting as a subtherapeutic dose that hastens the emergence of AMR [15,42].Unhygienic livestock and poultry farming in Bangladesh is a significant risk indicator for spreading zoonotic bacteria and antibiotic resistance to people and the environment [3].
Our data confirmed that AMR-B.cereus strains prevailed in all analyzed samples.The significance of this finding is underlined by earlier research [4,5,7,20,24,25] that showed Bacillus spp.can transfer ARGs.Bangladesh urgently requires the development of effective surveillance and control plans for the identification and prevention of ABR bacteria utilizing standard antibiotic susceptibility tests in regular animal and human microbiological laboratory settings.

Conclusion
It is the first investigation of the presence of ARGs of Bacillus spp. with public health significance in animal feed, food, and human stool in Bangladesh.The feedand food-borne Bacillus spp.exhibited species-specific trends in both phenotypic and genotypic resistance patterns with respect to antibiotic resistance.The associations of various antibiotic-resistant genes indicated a greater spread of β-lactamase, tetracycline, and erythromycin genes across the food chain.Animal feed and animal-derived products might serve as a channel for B. cereus propagation regarding their potential pathogenicity and the development of AMR in humans.This work validates the sources examined as major outlets for the spread of MDR bacteria and ARGs in the food chain of Bangladesh and once again highlights the urgency of a global campaign to combat AMR.

Minimum Bactericidal Concentration (MBC)
Five microliters of inoculum from the MIC experiment's well that had no bacterial growth after 24 h were spotted on NA (HiMedia, Mumbai, Maharashtra, India).The plates were incubated at 37 0 C for 16 to 24 h in order to evaluate the MBC described earlier [46].MBC was set as the lowest level on a NA plate where there was no visual growth.Bacillus spp. was detected, and it was determined that the growth of bacteria was bacteriostatic, while the absence of growth indicated bactericidal effects.
The NA plate was cultivated with the indicated inoculum dilution to test for contamination and cell viability [45].All analyses were performed in triplicate.

Determination of multidrug resistant (MDR) and multiple antibiotic resistance (MAR) index
MDR was considered to have at least one agent that was resistant to three or more types of antibiotics [47].

Resistance genotyping
The genomic DNA of various Bacillus species isolated from animal feed, food, and stool samples was extracted utilizing the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 (GW Vitek, Seoul, Korea).Following the manufacturer's instructions, pure colonies of Bacillus spp.isolates were prepared for DNA extraction from Tryptic Soy Broth (TSB) and Luria Bertani (LB) broth.A NanoDropTM 8000 spectrophotometer (Thermo Scientific, California, USA) was utilized to measure the concentration and purity of the eluted DNA.

Statistical analysis
The antibiotic susceptibility results were provided in MS-2016 Excel sheets and analyzed using IBM SPSS Statistics version 24 (SPSS Inc., Chicago, IL, USA).The prevalence was computed by a descriptive study and the Chi-square test, and the degree of significance was established using Pearson correlation coefficients.The statistical significance was calculated as * p < 0.05 and * * p < 0.01.

Table 4
Pearson correlation coefficients for pairs of antibiotics to assess antibiotic-resistant Bacillus isolates from animal feed, food and diarrhea *Correlation is significant at the 0.05 level (2-tailed), ** Correlation is significant at the 0.01 level (2 tailed), C Cannot be computed because at least one of the variables is constant, PG Penicillin G, AMC Amoxicillin-Clavulanic acid, CFM Cefixime, CTR Ceftriaxone, VAN Vancomycin, AZM Azithromycin, EM Erythromycin, TET Tetracycline, GEN Gentamicin, CM Clindamycin, NIT Nitrofurantoin, CIP Ciprofloxacin, LEV Levofloxacin, CMX Co-Trimoxazole

Table 6
Pearson correlation coefficients for pairs of ARGs of Bacillus isolates from animal feed, food and diarrhea ARGs Antibiotic resistance genes, *Correlation is significant at the 0.05 level (2-tailed), **Correlation is significant at the 0.01 level(2 tailed)

Table 7
profile of the isolated Bacillus spp

Table 7
(continued) MDR Multidrug resistance, PG Penicillin G, AMC Amoxicillin-Clavulanic acid, CFM Cefixime, CTR Ceftriaxone, VAN Vancomycin, AZM Azithromycin, EM Erythromycin, TET Tetracycline, CM Clindamycin, NIT Nitrofurantoin, CIP Ciprofloxacin, LEV Levofloxacin, CMX Co-Trimoxazole, NS Not significant The bacterial strains were cultivated aerobically in TSB at 37 °C with agitation at 225 rpm.The Ethical Reviewing Board on Institutional Animal Care and Use Committee at Noakhali Science and Technology University, Bangladesh, granted approval for the experimental protocols.An Informed Consent Form (ICF) was obtained prior to initiating research activities and collecting human stool samples.